Peptide nucleic acid oligomers (“PNA”) are nucleic acid analogs of DNA in which the sugar phosphodiester backbone has been replaced by a neutral, achiral polyamide (peptide) backbone having (2-aminoethyl) glycine carbonyl units linked to the purine or pyrimidine nucleobases through the glycine amino nitrogen and methylene carbonyl linkers (see, e.g., U.S. Pat. No. 5,539,082, the contents of which are incorporated herein by reference). PNAs have a high affinity for and hybridize to complementary RNA or DNA sequences by Watson-Crick base-pairing. Such hydridization is more rapid and more stable than hybridization between natural homoduplexes and possesses a greater potential for strand invasion because of the lack of electrostatic repulsion between the neutral PNA oligomers and the complementary RNA or DNA sequences. Furthermore, due to their altered backbone structures, PNAs are resistant to attack by nucleases and proteases. The chemical, physical and biological properties of PNAs make them useful, inter alia, as biomolecular tools, antisense and antigene agents, molecular probes and biosensors. For example, PNAs have been used to block protein expression on the transcriptional and translational level, monitor telomere length, inhibit human telomerase, affinity capture target nucleic acids, screen for genetic mutations, inhibit bacterial growth, and detect specific nucleic acid sequences in unamplified DNA.
In the case of antisense or antigene agents, one of the keys to their usefulness is that they must be taken up by the cells in a reasonable quantity so that they may reach their target in sufficient concentration. The uptake of oligonucleotides by cells, however, lacks efficiency. The uptake efficiency is particularly poor for PNAs because their neutral backbone linkages prevent effective transport across the cell membrane into the cytoplasm of the cell.
One of the known methods to improve PNA uptake into cells is to conjugate the PNA to a carrier molecule, such as DNA, cell-permeating peptide, peptidomimetic or other target molecule, to form a conjugate or chimera.
Oligonucleotide-peptide and PNA-peptide conjugates have been synthesized by sequential solid phase synthesis and by convergent synthesis in solution phase. Sequential solid phase synthesis of PNA-peptide conjugates is described by Simmons, C. G., Pitts, A. E., Mayfield, L. D., Shay, J. W., Corey, D. R. (1997) Bioorg. Med. Chem. Lett, 7, 3001–3006; Mayfield, L. D. and Corey, D. R. (1999) Anal. Biochchem., 268, 401–404; Branden, L. J., Mohamed, A. J., Smith, C. I. E. (1999) Nat. Biotech., 17, 784–787; Aldrian-Herrada, G., Desarmenien, M. G., Orcel, H., Boissin-Agasse, L., Mery, J., Brugidou, J., Rabie, A. (1998), Nucleic Acids Res., 26, 4910–4916; Basu, S. and Wickstrom, E. (1997) Bioconjugate Chem., 8, 481–488; Koch, T., et al. (1995) Tetrahedron Lett. 36, 6933–6936. Convergent synthesis in solution phase of PNA-peptide conjugates is described by Harrison, J. G., Frier, C., Laurant, R., Dennis, R., Raney, K. D., Balasubramanian, S. (1999) Bioorg. Med. Chem. Lett., 9, 1273–1278; and Pooga, M., et al. (1998) Nat. Biotech., 16, 857–861.
Both strategies have limited versatility and universal practical applicability. For the solid phase method, the outcome and quality of the synthesis of the conjugate are determined by the size and the solubility of the first entity on the solid support onto which the second entity is to be synthesized. For the solution phase conjugation method, separate step of purification of starting materials, synthesis and linking are required to form the conjugate. After conjugate formation, another purification step is required. Generally, the solution phase conjugation reaction is not quantitative and requires an excess of one of the entities to be conjugated. In some cases, the reaction requires a terminal cysteine to enable the formation of either a disulfide or thioether during the conjugation. Furthermore, no other cysteine units are permitted in the peptide.
Oligonucleotide-peptide conjugates have been prepared via the solid phase method using a bridging structure containing one protected hydroxy group for the oligonucleotide synthesis and one protected primary amino group for the peptide synthesis. See, for example, Juby, C. D., Richardson, C. D., Brousseau, R. (1991), Tetrahedron Lett. 32, 879–882; Basu, S., Wickstrom, E. (1995), Tetrahedron Lett., 36, 4943–4946; Antopolsky, M., Azhayev, A. (2000) Tetrahedron Lett. 41, 9113–9117; Antopolsky, M.; Azhayeva, E., Tengvall, U., Azhayev, A. (2002) Tetrahedron Lett. 43, 527–530. Unfortunately, this approach has limited applicability because the peptide must be synthesized before the oligonucleotide is synthesized and the appropriate choice of amino acid side chain protection must be made.
Thus, there remains a need to maintain the advantages of solid phase synthesis, inter alia, facile removal of reagents and the ability the automate the process, and improve the synthesis if both entities to be ultimately conjugated (e.g., oligonucleotide or PNA with a peptide) independently of one another, by minimizing or eliminating the influence of the first entity on the synthesis of the second entity. The crux of applicants' invention lies the use of a bridging unit attached to a solid support that permits conjugation of at least two entities, such as PNAs or oligonucleotides with peptides, wherein the influence of one entity on the synthesis or attachment of the other entity(ies) is minimized or eliminated. The use of the special bridging unit does not introduce chiral or pro-chiral centers and provides a high degree of conformational flexibility. The improved synthesis method will enable new conjugated oligomeric compounds to be prepared with enhanced therapeutic, prophylactic, research and diagnostic potential.